FIELD OF THE INVENTION
This invention relates generally to processes of purifying proteins and detecting residual ions in protein-containing, or other, solutions.
Decorin, also known as PG-II or PG-40, is a small proteoglycan produced by fibroblasts. Its core protein has a molecular weight of about 40,000 daltons. The core has been sequenced and it is known to carry a single glycosaminoglycan chain of the chondroitin sulfate/dermatan sulfate type. Most of the core protein of decorin is characterized by the presence of a leucine-rich repeat (LRR) of about 24 amino acids.
Proteoglycans are proteins that carry one or more glycosaminoglycan chains. The known proteoglycans carry out a wide variety of functions and are found in a variety of cellular locations. Many proteoglycans are components of extracellular matrix, where they participate in the assembly of matrix and effect the attachment of cells to the matrix.
Decorin has been used to prevent TGF.beta.-induced cell proliferation and extracellular matrix production. Decorin is therefore useful for reducing or preventing pathologies caused by TGF.beta.-regulated activity, such as cancer, glomerulonephritis and pathologies characterized by excess matrix. In cancer, for example, decorin can be used to destroy TGF.beta.-1's growth stimulating activity on the cancer cell. Decorin is also useful for reducing or inhibiting wound contraction, which involves proteins of the extracellular matrix.
Methods for expressing and purifying human recombinant decorin are known in the art, for example, as described in WO 90/00194. However, given the hydrophobic nature of decorin from the LRRs, more convenient and reproducible methods for purifying this proteoglycan from host cell contaminants have remained elusive.
After expressing human recombinant decorin, such as from decorin-expressing Chinese hamster ovary (CHO) cells, the proteoglycan can be substantially purified from the cell culture medium by the purification procedures of this invention. By using a combination of steps, and certain reagents, in particular a 2.4 to 3 molar GuHCl solution to elute decorin from a hydrophobic interactive column, the process of this invention provides a more convenient and reproducible process for purifying human recombinant decorin. To date, it provides the purest human recombinant decorin product known.
The present invention also provides a novel process for detecting the presence of guanidinium ions in a solution suspected of containing guanidinium ions, such as in protein-containing solutions where GuHCl has been used to purify the protein, as with the above purification scheme of decorin.
Guanidine is a buffer salt that is used in the purification of recombinant protein from the cellular milieu in the culture media. Residual guanidinium ions are presumed to be associated with the purified protein as a counter ion. Procedures have been designed to replace this counter ion with other ions, however, accurate determination of residual guanidinium ions in the low parts per million range is not available by current procedures, such as spectrophotometry. Residual guanidinium ion is considered to be a generally undesirable component in products designed for medicinal administration.
The invention provides an ion chromatographic method which uses a conductivity detector to detect and quantify guanidinium ions present in a solution. The method is highly sensitive, reproducible, and fast. The high sensitivity to guanidinium ions, in the parts per million range, is remarkable in the presence of an overwhelming concentration of sodium. The process of this invention also has the advantage of requiring only minimal sample volumes and can be accomplished with very little sample preparation.